A Monte Carlo study of the amylosic chain conformation

Monte Carlo studies of the unperturbed amylosic chain conformation have been carried out in the approximation of separable chain configuration energies. Sample chains of arbitrary chain length have been generated so as to be distributed consistent with refined estimates of the configuration energy and thus suitable for evaluation of averages of the desired configuration-dependent properties. Perspective drawings of representative chains from the Monte Carlo sample have been made for comparison with standard idealizations of amylosic chain conformation. He molecular model employed generates a randomly coiling chain possessing perceptible regions of left-handed pseudohelical backbone trajectory. Distribution functions for the end-to-end distance of short amylosic chains disclose some propensity for the chain to suffer self-intersections at sort range in the chain sequence, which may vitiate the usual amylosic chain models based on the assumed independence of sets of glycosidic linkage torsion angles. The amylosic persistence vector and persistence length have been calculated as a function of chain length for the chain model employed.     

Conformational behavior of the polysaccharide backbone of murein

The energetically possible conformations for the alternating heteropolysaccharide backbone of murein, consisting of N-acetylglucosamine and N-acetylmuramic acid, were calculated using an empirical approach. The calculations were carried out for regular as well as for random-chain polymers, resulting in a model for the saccharide strands featuring extended chains with a length increment of 0.98–1.02 nm per disaccharide unit and peptide attachment sites at every second saccharide residuum pointing into all directions with propagation angles of 80–100° between consecutive sites.     

Proline-Rich Salivary Proteins Have Extended Conformations

Three basic proline-rich salivary proteins have been produced through the recombinant route. IB5 is a small basic proline-rich protein that is involved in the binding of plant tannins in the oral cavity. II-1 is a larger protein with a closely related backbone; it is glycosylated, and it is also able to bind plant tannins. II-1ng has the same polypeptidic backbone as II-1, but it is not glycosylated. Small angle x-ray scattering experiments on dilute solutions of these proteins confirm that they are intrinsically disordered. IB5 and II-1ng can be described through a chain model including a persistence length and cross section. The measured radii of gyration (R_g = 27.9 and 41.0 ± 1 Å respectively) and largest distances (r_max = 110 and 155 ± 10 Å respectively) show that their average conformations are rather extended. The length of the statistical segment (twice the persistence length) is b = 30 Å, which is larger than the usual value (18 Å − 20 Å) for unstructured polypeptide chains. These characteristics are presumably related to the presence of polyproline helices within the polypeptidic backbones. For both proteins, the radius of gyration of the chain cross-section is R_c = 2.7 ± 0.2Å. The glycosylated protein II-1 has similar conformations but the presence of large polyoside sidegroups yields the structure of a branched macromolecule with the same hydrophobic backbone and hydrophilic branches. It is proposed that the unusually extended conformations of these proteins in solution facilitate the capture of plant tannins in the oral cavity.     

An algorithm for determining the conformation of polypeptide segments in proteins by systematic search

The feasibility of determining the conformation of segments of a polypeptide chain up to six residues in length in globular proteins by means of a systematic search through the possible conformations has been investigated. Trial conformations are generated by using representative sets of phi, psi, and chi angles that have been derived from an examination of the distributions of these angles in refined protein structures. A set of filters based on simple rules that protein structures obey is used to reduce the number of conformations to a manageable total. The most important filters are the maintenance of chain integrity and the avoidance of too-short van der Waals contacts with the rest of the protein and with other portions of the segment under construction. The procedure is intended to be used with approximate models so that allowance is made throughout for errors in the rest of the structure. All possible main chains are first constructed and then all possible side-chain conformations are built onto each of these. The electrostatic energy, including a solvent screening term, and the exposed hydrophobic area are evaluated for each accepted conformation. The method has been tested on two segments of chain in the trypsin like enzyme from Streptomyces griseus. It is found that there is a wide spread of energies among the accepted conformations, and the lowest energy ones have satisfactorily small root mean square deviations from the X-ray structure.     

Conformational dynamics of unfolded peptides as a function of chain length: a frequency domain fluorescence approach

The fluorescence emission decays of single-tryptophan-containing peptides of different chain lengths in their unfolded state were investigated in the frequency domain. The data were analyzed using different functions, i.e., exponential fit and probability-density functions of different shape. We found that unimodal Lorentzian distributions best describe the fluorescence decays. This finding agrees with the point of view, now broadly accepted, that rapid motions exist in polypeptides. As a consequence of this flexibility, a large variety of conformations, with an unequal perturbation of tryptophan in its excited state, is generated. The lifetime distribution center was independent of the length of the polypeptide chain but strongly related to the nature of the amino acid residues located in the proximity of the tryptophan in the primary structure. The full width at half maximum, W, of the lifetime distribution was found to be related to the length of unfolded polypeptide by the empirical logarithmic relationship W = 0.83 log n, where n indicates the number of residues. For short peptides, a single lifetime or a narrow range of lifetimes is observed because of the fast relaxation of the tryptophanyl environment. On peptide lengthening, the spectrum of conformations, which the peptide can assume, increases; this causes a complex fluorescence decay represented by a lifetime distribution. For long polypeptide chains, the motions of the regions far from tryptophan do not significantly perturb the chromophore environment.     

Extended N-Sulfated Domains Reside at the Nonreducing End of Heparan Sulfate Chains

Heparan sulfate (HS) serves as a cell-surface co-receptor for growth factors, morphogens, and chemokines. These HS and protein binding events depend on the fine structure and distribution of domains along an HS chain. A given domain can vary in terms of uronic acid epimer, N- and O-sulfate, and N-acetate content. The most highly sulfated regions of HS chains, N-sulfated (NS) domains, play prominent roles in HS and protein binding. We have analyzed HS oligosaccharides from various mammalian sources and provide evidence that NS domains residing at the nonreducing end (NRE) are, on average, longer than those residing in the internal regions of the chain. Additionally, they are more highly sulfated than their internal counterparts. These features are independent of the sulfation pattern of the bulk HS chains. From disaccharide analysis, it is clear that NS domains do not always occupy HS NREs. However, when they do, they tend to terminate in a subset of N-sulfated disaccharides. Our observations are consistent with a significant role of NRE NS domains in HS-growth factor interactions.     

Constructing side chains on near-native main chains for ab initio protein structure prediction

Is there value in constructing side chains while searching protein conformational space during an ab initio simulation? If so, what is the most computationally efficient method for constructing these side chains? To answer these questions, four published approaches were used to construct side chain conformations on a range of near-native main chains generated by ab initio protein structure prediction methods. The accuracy of these approaches was compared with a naive approach that selects the most frequently observed rotamer for a given amino acid to construct side chains. An all-atom conditional probability discriminatory function is useful at selecting conformations with overall low all-atom root mean square deviation (r.m.s.d.) and the discrimination improves on sets that are closer to the native conformation. In addition, the naive approach performs as well as more sophisticated methods in terms of the percentage of chi(1) angles built accurately and the all-atom r. m.s.d., between the native and near-native conformations. The results suggest that the naive method would be extremely useful for fast and efficient side chain construction on vast numbers of conformations for ab initio prediction of protein structure.     

Reduced Expression of EXTL2, a Member of the Exostosin (EXT) Family of Glycosyltransferases,in Human Embryonic Kidney 293 Cells Results in Longer Heparan Sulfate Chains

Heparan sulfate proteoglycans are ubiquitously located on cell surfaces and in the extracellular matrices. The negatively charged heparan sulfate chains interact with a multitude of different proteins, thereby influencing a variety of cellular and developmental processes, for example cell adhesion, migration, tissue morphogenesis, and differentiation. The human exostosin (EXT) family of genes contains five members: the heparan sulfate polymerizing enzymes, EXT1 and EXT2, and three EXT-like genes, EXTL1, EXTL2, and EXTL3. EXTL2 has been ascribed activities related to the initiation and termination of heparan sulfate chains. Here we further investigated the role of EXTL2 in heparan sulfate chain elongation by gene silencing and overexpression strategies. We found that siRNA-mediated knockdown of EXTL2 in human embryonic kidney 293 cells resulted in increased chain length, whereas overexpression of EXTL2 in the same cell line had little or no effect on heparan sulfate chain length. To study in more detail the role of EXTL2 in heparan sulfate chain elongation, we tested the ability of the overexpressed protein to catalyze the in vitro incorporation of N-acetylglucosamine and N-acetylgalactosamine to oligosaccharide acceptors resembling unmodified heparan sulfate and chondroitin sulfate precursor molecules. Analysis of the generated products revealed that recombinant EXTL2 showed weak ability to transfer N-acetylgalactosamine to heparan sulfate precursor molecules but also, that EXTL2 exhibited much stronger in vitro N-acetylglucosamine-transferase activity related to elongation of heparan sulfate chains.     

Solution conformations of pectin polysaccharides: Determination of chain characteristics by small angle neutron scattering,viscometry, and molecular modeling

The solution behavior of pectin polysaccharides has been investigated by small angle neutron scattering (SANS), viscosimetric, and molecular modeling studies. The samples used in the experimental study were obtained from apple and citrus and had degrees of methylation ranging from 28 to 73%, with a rhamnose content lying between 0.6 and 2.2%. Persistence lengths, derived from intrinsic viscosity measurements, ranged from 59 to 126 Å, whereas those derived by SANS were between 45 and 75 Å. These values correspond to 10–17 monomer units. The modeling simulations were performed for both homogalacturonan itself and homogalacturonan carrying various degrees of rhamnose inserts (rhamnogalacturonan). This required the evaluation of the accessible conformational space for the eight disaccharides that represent the constituent repeating segments of the homogalacturonan and rhamnogalacturonan polysaccharides. For each dimer, complete conformational analysis was accomplished using the flexible residue method of the MM3 molecular mechanics procedure and the results used to access the configurational statistics of representative pectic polysaccharide chains. For homogalacturonan, an extended chain conformation having a persistence length of 135 Å (corresponding to 30 monomers) was predicted. The inclusion of varying amounts of rhamnose units (5–25%) in the model in strict alternating sequence with galacturonate residues (equivalent to the rhamnogalacturonan “hairy region” chains) only slightly reduced the calculated persistence length. The extended overall chain conformation remained relatively unchanged as a consequence of the self-cancellation of the kinking effects of successive paired rhamnose units. © 1996 John Wiley & Sons, Inc.     

The nucleic acid distribution function: evaluation of the loop closure probability

We present a detailed theoretical study of nucleic acid distance distribution functions for chain lengths up to thirty-five nucleotides. The distribution functions were found by the Monte Carlo techniques described previously and, where they are symmetric, by reconstruction from their even moments. A comparison of the two approaches allows an assessment of the utility of expansion procedures for stiff chains (C_∞ ≈ 17), as a function of length and extension. It was found that for chains with fewer than fourteen nucleotides over seventy even moments were required to obtain a reliable function for extensions R ≥ (〈R²〉)^1/2. On the other hand at thirty-five nucleotides the first ten even moments were sufficient to reconstruct the distribution for all but extreme extensions. The other feature of the calculation is the prediction of a loop weighting function. It was found that for a thirty-five nucleotide chain, the form of the distribution function is very nearly gaussian for R < (〈R²〉)^1/2. Consequently a loop weighting function based on detailed crystallographic data is now known for all values of N. The first derivative of the function is within 8% of the Jacobson-Stockmayer value at 170 nucleotides, but differs severely from the latter for chains with less than twenty nucleotides.     

Cotranslational folding promotes beta-helix formation and avoids aggregation in vivo

Newly synthesized proteins must form their native structures in the crowded environment of the cell, while avoiding non-native conformations that can lead to aggregation. Yet, remarkably little is known about the progressive folding of polypeptide chains during chain synthesis by the ribosome or of the influence of this folding environment on productive folding in vivo. P22 tailspike is a homotrimeric protein that is prone to aggregation via misfolding of its central β-helix domain in vitro. We have produced stalled ribosome:tailspike nascent chain complexes of four fixed lengths in vivo, in order to assess cotranslational folding of newly synthesized tailspike chains as a function of chain length. Partially synthesized, ribosome-bound nascent tailspike chains populate stable conformations with some native-state structural features even prior to the appearance of the entire β-helix domain, regardless of the presence of the chaperone trigger factor, yet these conformations are distinct from the conformations of released, refolded tailspike truncations. These results suggest that organization of the aggregation-prone β-helix domain occurs cotranslationally, prior to chain release, to a conformation that is distinct from the accessible energy minimum conformation for the truncated free chain in solution.     

Enumerating Designing Sequences in the HP Model

The hydrophobic/polar HP model on the square lattice has been widely used toinvestigate basics of protein folding. In the cases where all designing sequences (sequences with unique ground states) were enumerated without restrictions on the number of contacts, the upper limit on the chain length N has been 18–20 because of the rapid exponential growth of thenumbers of conformations and sequences. We show how a few optimizations push this limit by about 5 units. Based on these calculations, we study the statistical distribution of hydrophobicity along designing sequences. We find that the average number of hydrophobic and polar clumps along the chains is larger for designing sequences than for random ones, which is in agreement with earlier findings for N 18 and with results for real enzymes. We also show that this deviation from randomness disappears if the calculations are restricted to maximally compact structures.     

The spatial distributions of randomly coiling polynucleotides

Statistical mechanical averages of vectors and tensors characterizing the allowed configurations of randomly coiling polynucleotides have been calculated for chains of 2⁰–2¹⁰ repeating units. Specifically, the persistence vector p = 〈 r 〉 has been obtained as a function of chain length. Configurational averages of the Cartesian tensors formed from the displacement vector ρ = r – p have been computed up to and including the tensor of seventh rank. From these tensors the three-dimensional spatial distributions of end-to-end vectors have been constructed to provide comprehensive pictures of the directional tendencies of the randomly coiling polynucleotide. The elements of the third and fourth moment tensors, however, give sufficient information to represent accurately the qualitative features of the spatial distributions. For long chains, more than 2⁶ (64) repeating units, the spatial distributions assume spherically symmetric shapes that can be adequately characterized by one-dimensional radial distribution functions. These radial distribution functions agree well with the radial distributions calculated from Monte Carlo samples containing more than 5000 chains. The constraints of fixed bond lengths, fixed bond angles, and hindered internal rotations severely distort the spatial distributions of short polynucleotide chains to mushroom-shaped volumes. These skewed distributions provide information useful to the analysis of small, single-stranded loops, bulges, and circles. The formation of these structures requires the termini of the polynucleotides to lie within specifically delineated volumes with respect to coordinate systems affixed to the first bonds of the chains. The extent to which these loop closure volumes overlap the three-dimensional spatial distributions provides estimates of loop formation that are much more reliable than earlier studies based upon the radial distribution function.     

Liver transduction with recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype

We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 x 10(10) to 1.8 x 10(12) particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in approximately 80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.     

The chain length of the glycans in bacterial cell walls (Short Communication)

The glycan chain length of peptidoglycan was measured by reduction with NaB³H₄ and isolation of the resulting muramitol, indicative of the length of the chains as biosynthesized, and glucosaminol, which measured the length of the chains after rupture by endo-β-N-acetylglucosaminidases. Measurement of the non-reducing terminal N-acetylglucosamine by Smith degradation confirmed the result.     

Effects of denaturants on the dynamics of loop formation in polypeptides

Quenching of the triplet state of tryptophan by close contact with cysteine has been used to measure the reaction-limited and diffusion-limited rates of loop formation in disordered polypeptides having the sequence cys-(ala-gly-gln)j-trp (j=1-9). The decrease in the length-dependence of the reaction-limited rate for short chains in aqueous buffer, previously attributed to chain stiffness, is not observed at high concentrations of chemical denaturant (6 M GdmCl and 8 M urea), showing that denaturants increase chain flexibility. For long chains, both reaction-limited and diffusion-limited rates are significantly smaller in denaturant and exhibit a steeper length dependence. The results can be explained using end-to-end distributions from a wormlike chain model in which excluded volume interactions are incorporated by associating a 0.4-0.5 nm diameter hard sphere with the end of each virtual peptide bond. Fitting the data with this model shows that the denaturants reduce the persistence length from approximately 0.6 nm to approximately 0.4 nm, only slightly greater than the length of a peptide bond. The same model also describes the reported length dependence for the radii of gyration of chemically denatured proteins containing 50-400 residues. The end-to-end diffusion coefficients obtained from the diffusion-limited rates are smaller than the sum of the monomer diffusion coefficients and exhibit significant temperature dependence, suggesting that diffusion is slowed by internal friction arising from barriers to backbone conformational changes.     

Computational simulation of the statistical properties of unfolded proteins

A simple Monte Carlo method was used to generate ensembles of simulated polypeptide conformations that are restricted only by steric repulsion. The models used for these simulations were based on the sequences of four real proteins, ranging in size from 26 to 268 amino acid residues, and included all non-hydrogen atoms. Two sets of calculations were performed, one that included only intra-residue steric repulsion terms and those between adjacent residues, and one that included repulsion terms between all possible atom pairs, so as to explicitly account for the excluded volume effect. Excluded volume was found to increase the average radius of gyration of the chains by 20-40%, with the expansion factor increasing with chain length. Contrary to recent suggestions, however, the excluded volume effect did not greatly restrict the distribution of dihedral angles or favor native-like topologies. The average dimensions of the ensembles calculated with excluded volume were consistent with those measured experimentally for unfolded proteins of similar sizes under denaturing conditions, without introducing any adjustable scaling factor. The simulations also reproduced experimentally determined effective concentrations for the formation of disulfide bonds in reduced and unfolded proteins. The statistically generated ensembles included significant numbers of conformations that were nearly as compact as the corresponding native proteins, as well as many that were as accessible to solvent as a fully extended chain. On the other hand, conformations with as much buried surface area as the native proteins were very rare, as were highly extended conformations. These results suggest that the overall properties of unfolded proteins can be usefully described by a random coil model and that an unfolded polypeptide can undergo significant collapse while losing only a relatively small fraction of its conformational entropy.     

Further studies on the peptido-galactomannan from the yeast form of Cladosporium werneckii. Identification of O-acetyl substituents and isolation of the peptide components following beta-elimination.

The pathogenic yeast Cladosporium werneckii produces a surface peptido-phosphogalactomannan (PPGM) with a peptide backbone rich in serine and threonine to which three types of carbohydrate chains are linked. These chains are: Type a, glactomannan units linked through phosphodiester bonds to produce long chains of molecular weight about 50,000; type b more numerous short mannosyl oligosaccharide units, and type c, more infrequent, long galactomannan chains. The first two are linked to the peptide through alkali-labile bonds to serine and threonine of the peptide whereas type c chains are linked through alkali-stable bonds (Lloyd, K. O. (1972) Biochemistry, 11, 3884–3890). The PPGM sample can be separated into three or four major components by diethylaminoethyl (DEAE-) Sephadex chromatography. By means of sequential degradations with alkali and acid, the structural basis for this heterogeneity has been demonstrated. It is due to the presence of different proportions of the three types of chains in the various fractions. The presence of O-acetyl groups, mainly on the a chains, was demonstrated in PPGM by chemical analysis and by proton and ¹³C nuclear magnetic resonance spectroscopy. Purified a chains were isolated from PPGM following Pronase digestion. By taking advantage of the alkali lability of the carbohydrate-protein linkages it has been possible to cleave the peptide moiety away from the carbohydrate. By sequential chromatography on Bio-Gel P-100, Dowex 1 and Bio-Gel P-100, five modified peptide fractions were isolated. The molecular weights of these fractions varied from 9,500 to 18,500 as judged by polyacrylamide-gel electrophoresis. Unlike the original peptide, the modified peptides contained low amounts of serine and threonine. The predominant amino acids were alanine, glycine, aspartic acid and glutamic acid which together make up between 51 and 55% of the peptides. The high content of the last two amino acids accounts for the acidic nature of the peptides. It appears that each fraction consists of a slightly heterogeneous population of peptides very similar in amino acid composition. It is not clear whether the compositional and size heterogeneity exists in the original peptide or whether it arose during the isolation procedure.     

Determination of the conformation of folding initiation sites in proteins by computer simulation

Experimental evidence and theoretical models both suggest that protein folding begins by specific short regions of the polypeptide chain intermittently assuming conformations close to their final ones. The independent folding properties and small size of these folding initiation sites make them suitable subjects for computational methods aimed at deriving structure from sequence. We have used a torsion space Monte Carlo procedure together with an all-atom free energy function to investigate the folding of a set of such sites. The free energy function is derived by a potential of mean force analysis of experimental protein structures. The most important contributions to the total free energy are the local main chain electrostatics, main chain hydrogen bonds, and the burial of nonpolar area. Six proposed independent folding units and four control peptides 11–14 residues long have been investigated. Thirty Monte Carlo simulations were performed on each peptide, starting from different random conformations. Five of the six folding units adopted conformations close to the experimental ones in some of the runs. None of the controls did so, as expected. The generated conformations which are close to the experimental ones have among the lowest free energies encountered, although some less native like low free energy conformations were also found. The effectiveness of the method on these peptides, which have a wide variety of experimental conformations, is encouraging in two ways: First, it provides independent evidence that these regions of the sequences are able to adopt native like conformations early in folding, and therefore are most probably key components of the folding pathways. Second, it demonstrates that available simulation methods and free energy functions are able to produce reasonably accurate structures. Extensions of the methods to the folding of larger portions of proteins are suggested. © 1995 Wiley-Liss, Inc.     

Cotranslational Folding Increases GFP Folding Yield

Protein sequences evolved to fold in cells, including cotranslational folding of nascent polypeptide chains during their synthesis by the ribosome. The vectorial (N- to C-terminal) nature of cotranslational folding constrains the conformations of the nascent polypeptide chain in a manner not experienced by full-length chains diluted out of denaturant. We are still discovering to what extent these constraints affect later, posttranslational folding events. Here we directly address whether conformational constraints imposed by cotranslational folding affect the partitioning between productive folding to the native structure versus aggregation. We isolated polyribosomes from Escherichia coli cells expressing GFP, analyzed the nascent chain length distribution to determine the number of nascent chains that were long enough to fold to the native fluorescent structure, and calculated the folding yield for these nascent chains upon ribosome release versus the folding yield of an equivalent concentration of full-length, chemically denatured GFP polypeptide chains. We find that the yield of native fluorescent GFP is dramatically higher upon ribosome release of nascent chains versus dilution of full-length chains from denaturant. For kinetically trapped native structures such as GFP, folding correctly the first time, immediately after release from the ribosome, can lead to lifelong population of the native structure, as opposed to aggregation.